U.S. patent application number 17/379496 was filed with the patent office on 2022-01-20 for method and system for virus and protein-antibody interactions detection and monitoring based on optical light intensity and electrical parameters.
The applicant listed for this patent is United Arab Emirates University. Invention is credited to Mahmoud F.Y. Al Ahmad, Farah Mustafa, Tahir A. Rizvi.
Application Number | 20220018839 17/379496 |
Document ID | / |
Family ID | |
Filed Date | 2022-01-20 |
United States Patent
Application |
20220018839 |
Kind Code |
A1 |
Al Ahmad; Mahmoud F.Y. ; et
al. |
January 20, 2022 |
Method And System For Virus And Protein-Antibody Interactions
Detection And Monitoring Based On Optical Light Intensity And
Electrical Parameters
Abstract
A novel method of detecting and destroying viral transmissions
such as SARS-CoV-2 transmission is described. The proposed
technique uses a light source such as that from a smart phone and a
mobile spectrophotometer to enable detection of proteins in
solution. The technique allows for detecting soluble preparations
of for example spike protein subunits from SARS-CoV-2, followed by
detection of the actual binding potential of the spike protein with
its host receptor, for example the angiotensin-converting enzyme 2
(ACE2) or other antigens or elements. The results are validated by
showing that this method can detect antigen-antibody binding using
two independent protein-antibody pairs. Finally, this technique is
combined with DC bias to show that introduction of a current in the
system can be used to disrupt the antigen-antibody reaction,
suggesting that this technique can be a powerful means of
disrupting virus transmission by destroying virus-receptor
interactions.
Inventors: |
Al Ahmad; Mahmoud F.Y.; (Al
Ain, AE) ; Rizvi; Tahir A.; (Al Ain, AE) ;
Mustafa; Farah; (Al Ain, AE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
United Arab Emirates University |
Al Ain |
|
AE |
|
|
Appl. No.: |
17/379496 |
Filed: |
July 19, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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63054022 |
Jul 20, 2020 |
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International
Class: |
G01N 33/569 20060101
G01N033/569; G01N 21/47 20060101 G01N021/47 |
Claims
1. A method of opto-electrical detection of the presence of
possible bindings or interactions between analytes in a sample,
comprising: exposing a sample to light from a light source;
detecting light passing through the sample; applying an electrical
field over the sample; determining the values of a selection of
light scattering parameters for the light passing through the
sample in response to the electrical field; and/or determining the
values of a selection of electrical parameters, such as electrical
scattering parameters, in response to the electrical field;
determining the presence of bindings or interactions between
analytes in the sample based on the values of the determined light
scattering parameters and/or the determined values of the
electrical parameters, for example electrical scattering
parameters.
2. The method of claim 1, wherein the presence of bindings or
interactions between analytes in the sample is determined based on
a predetermined characteristic of the detected light scattering
parameters, and/or electrical scattering parameters, for specific
values of the electric parameters.
3. (canceled)
4. (canceled)
5. (canceled)
6. The method of claim 1, wherein a selection of one or more light
scattering parameters, for example intensities, and/or electrical
scattering parameters are measured and possibly recorded over a
time-period and/or mapped to a time domain.
7. (canceled)
8. (canceled)
9. (canceled)
10. (canceled)
11. The method of claim 1, wherein: the sample is added to a holder
or container aligned with the light source and the light detector
such that measurement of light passing through the sample is
enabled; and measured responses in the form of the determined
values of said selection of optical or electrical parameters in
response to the electrical field are collected to determine the
presence of bindings or interactions between analytes in the
sample.
12. The method of 11, wherein collecting measured responses
comprises one or more of: collecting directly measured light
intensity; and/or collecting directly measured light intensity and
establishing direct relationship with time; and/or processing
direct measured data to extract parameters or a set of parameters
and correlating said parameters with time to establish relationship
with the presence of bindings or interactions between analytes in
the sample.
13. (canceled)
14. The method of claim 1, wherein a mathematical relation for the
determined parameters represents the process of bindings or
interactions between analytes in the sample.
15. (canceled)
16. (canceled)
17. (canceled)
18. (canceled)
19. The method of claim 1, wherein the presence and/or the process
of one or more of said bindings or interactions is determined,
detected and/or monitored based on measurements of an electrical
parameter, for example an electrical scattering parameter, for
example one or more of the voltage, the current, the capacitance
and/or the impedance over time in relation to a said applied
electric field.
20. (canceled)
21. (canceled)
22. (canceled)
23. The method of claim 1, used for virus detection and comprising
collecting and processing electrical and optical responses
individually or simultaneously to extract a set of parameters for
detection, quantification and identification of virus.
24. (canceled)
25. The method of claim 23, further being adapted and used for
detection of one or more of virus from the group SARS-CoV-2, SARS,
MERS, influenza, respiratory syncytial virus (RSV), adenoviruses,
or any other respiratory virus.
26. The method of claim 23, further comprising applying an
electrical pulse over or through a sample containing analytes from
a test subject and selected antibodies, thereby enabling
electro-insertion of said antibodies into any virus cell present in
the sample.
27. (canceled)
28. The method of claim 23, wherein a characteristic of the
determined light scattering parameters, and/or electrical
scattering parameters, for specific values of the electric
parameters is determined to measure the viral nucleocapsid protein
and anti-N antibody interactions in a sample to differentiate
between SARS-CoV-2 negative and positive nasal swab samples.
29. (canceled)
30. The method of claim 1, wherein: a said sample is placed and
distributed in one or more microfluidic channels; measurement of
said light scattering parameters and/or said electrical parameters
is conducted for the sample content in each of said microfluidic
channels; and virus concentration and/or virus load is determined
based on said measured parameters.
31. (canceled)
32. The method of claim 30, wherein: said sample is placed and
distributed in a plurality of parallel microfluidic channels; the
antibody content is serially diluted in said plurality of parallel
channels, simultaneously measuring said parameters in said
plurality of channels; and said virus concentration and/or virus
load is determined based on said measured parameters.
33. (canceled)
34. A system of opto-electrical detection of the presence of
possible bindings or interactions between analytes in a sample,
comprising: a light source configured to emit or transfer natural
or manmade light and to expose a sample with said light; an
electric field device configured to apply a biasing electric field
over the sample; a light detector configured to detect light
passing through a said light exposed sample; an electric parameter
detector configured to detect electric parameters; a processing
device having code portions configured to direct the processor to:
determine the values of a selection of one or more light scattering
parameters of the detected light passing through a said light
exposed sample; determine the values of a selection of electrical
parameters, for example electric scattering parameters, in response
to the electrical field, and to determine the presence of bindings
or interactions between analytes in the sample based on the values
of the determined light scattering parameters, and/or electric
scattering parameters, and the determined values of the electrical
parameters.
35. The system of claim 30, wherein the processing device comprises
code portions configured to determine the presence of bindings or
interactions between analytes in the sample based on a
predetermined characteristic of the detected light scattering
parameters, and/or detected electrical scattering parameters, for
specific values of the electric parameters.
36. (canceled)
37. (canceled)
38. (canceled)
39. (canceled)
40. (canceled)
41. The system of claim 34, further comprising: a transparent
holder or container allowing light to pass through the material
without appreciable scattering of light and being configured for
holding a sample, for example on or more of a plate, box, tube or
any type of transparent paper or other transparent material.
42. (canceled)
43. (canceled)
44. The system of claim 34, wherein the electric field device
comprises two electrodes configured or configurable at respective
sides of a sample and being couplable to an electric energy
source.
45. (canceled)
46. (canceled)
47. (canceled)
48. The system of claim 34, wherein the processing device comprises
code portions configured to apply a mathematical relation for the
determined parameters to represent the process of bindings or
interactions between analytes in the sample.
49. (canceled)
50. (canceled)
51. (canceled)
52. (canceled)
53. The system of claim 34, wherein the processing device comprises
code portions configured to determine, detect and/or monitor the
presence and/or the process of one or more of said bindings or
interactions based on measurements of an electrical parameter, for
example an electrical scattering parameter, for example one or more
of the voltage, the current, the capacitance and/or the impedance
over time in relation to a said applied electric field.
54. (canceled)
55. (canceled)
56. (canceled)
57. The system of claim 34, wherein the processing device comprises
code portions configured to collecting and processing electrical
and optical responses individually or simultaneously to extract a
set of parameters for detection, quantification and identification
of virus.
58. (canceled)
59. The system of claim 57, further being adapted and used for
detection of one or more of virus from the group SARS-CoV-2, SARS,
MERS, influenza, respiratory syncytial virus (RSV), adenoviruses,
or any other respiratory virus.
60. The system of claim 57, further being configured to apply an
electrical pulse over or through a sample containing analytes from
a test subject and selected antibodies, thereby enabling
electro-insertion of said antibodies into any virus cell present in
the sample.
61. (canceled)
62. The system of claim 57, wherein the processing device comprises
code portions configured to determine a characteristic of the light
scattering parameters, and/or an electrical scattering parameter,
for specific values of the electric parameters and to measure the
viral nucleocapsid protein and anti-N antibody interactions in a
sample to differentiate between SARS-CoV-2 negative and positive
nasal swab samples.
63. The system of claim 57, further comprising: a sample strip
configured for collecting a sample of analytes from a test object,
the sample strip having an electrode coated thereon and configured
to conduct current measurements directly, and said sample strip
comprising a portion coated with antibodies.
64. The system of claim 63, wherein the sample strip is coated with
an antibody configured to bind or interact with of one or more of
virus from the group SARS-CoV-2, SARS, MERS, influenza, respiratory
syncytial virus (RSV), adenoviruses, or other respiratory
virus.
65. (canceled)
66. The system of claim 57, further comprising: a device for nasal
sampling configured such that a sampling strip is attachable at a
section of the nasal sampling device that is configured to be
insertable in the nose of a human test object, the nasal sampling
device comprising two electrodes configured to apply an electric
field over a sample of analytes collected on a said sampling
strip.
67. The system of claim 57, wherein the nasal sampling device
further comprises an integrated light source and detector
configured to expose the sample to light and to detect light
passing through the sample.
68. The system of claim 57, configured such that when the presence
of a binding or interaction between a virus and an antibody is
detected in a sample from a patient, an indication signal is
presented confirming that patient is infected with the tested
virus.
69. (canceled)
70. The system of claim 34, comprising: a microfluidic sensing
device with one or more one or more microfluidic channels
configured for placing and distributing a said sample in one or
more of said microfluidic channels; one or more sensors configured
for measurement of said light scattering parameters and/or said
electrical parameters for the sample content in each of said
microfluidic channels; and code portions, in said processing
device, configured to determine virus concentration and/or virus
load based on said measured parameters.
71. (canceled)
72. The system of claim 70, wherein: said microfluidic sensing
device is configured with a plurality of parallel microfluidic
channels for placing and distributing said sample; said
microfluidic sensing device is configured for serially diluting the
antibody content in said plurality of parallel channels,
simultaneously measuring said parameters in said plurality of
channels; and said system is configured to determine said virus
concentration and/or virus load based on said measured
parameters.
73. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a U.S. Non-Provisional Patent
Application that claims priority to U.S. Provisional Patent
Application No. 63/054,022, filed on Jul. 20, 2020 and entitled
"Method and System for Virus and Protein-Antibody Interactions
Detection and Monitoring Based on Optical Light Intensity and
Electrical Parameters", the entirety of which is hereby
incorporated by reference.
FIELD OF THE INVENTION
[0002] The present disclosure relates in general to a method and
system for virus detection and monitoring, and more specifically to
a method and system for virus and protein-antibody interactions
detection and monitoring based on optical light intensity and
electrical parameters.
BACKGROUND
[0003] In late December 2019, patients with an atypical pneumonia
due to a novel coronavirus were reported in Wuhan, China. Since
then, the novel coronavirus disease 2019 (COVID-19) has become a
pandemic that has spread worldwide to virtually every country. This
pandemic has caused massive social and economic disruptions in
nearly every country and therefore global research and development
efforts are being geared towards development of vaccines and
therapeutics for the prevention and treatment of COVID-19, in order
to normalize the situation.
[0004] Unfortunately, the complete clinical picture of COVID-19 is
not yet fully known and most likely depends upon a number of
factors, including virus characteristics. As of Jul. 7, 2020, more
than 11,645,109 cases of COVID-19 infection had been confirmed
worldwide with 538,780 deaths, revealing a case fatality rate (CFR)
of 4.6%. Successful detection of SARS coronavirus 2 (SARS-CoV-2)
plays an important role in stopping the spread.
[0005] The novel severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) that causes COVID-19 enters the susceptible cells
primarily via endocytosis using its spike (S) protein (3-5). The
viral S protein is a homotrimer that protrudes from the virion
surface (6) and is responsible for entry into susceptible cells by
binding to the human angiotensin converting enzyme 2 (ACE2) protein
(3-5). Once internalized, the virus starts to replicate within the
cell (7). The nucleocapsid (N) protein of SARS-CoV-2, is the
largest structural protein of the virus which coats its large
genomic RNA and is responsible for creating its helical structure
(8). Compared to the viral S protein, the N protein is much more
conserved (.about.90%), is expressed at high levels during
infection, and is highly immunogenic (8). This is for example
described in the publication Y. Cong, M. Ulasli, H. Schepers, M.
Mauthe, P. V'kovski, F. Kriegenburg, V. Thiel, C. A. M. de Haan, F.
Reggiori, Nucleocapsid Protein Recruitment to
Replication-Transcription Complexes Plays a Crucial Role in
Coronaviral Life Cycle. J. Virol. 94 (2019),
doi:10.1128/JVI.01925-19.
[0006] Currently, oropharyngeal and nasopharyngeal swabs are
primarily used for virus detection. However, it is not clear how
many virus particles of SARS-CoV-2 are needed to trigger an
infection. It has been anticipated that the corresponding dose to
establish an infection in exposed people could be as little as 10
virus particles. Other studies suggest a relatively higher dose,
ranging from a few hundred to thousands of particles. It has been
estimated that SARS-CoV-2 exhibits a higher rate of virus
replication compared to SARS-CoV-1 which can increase disease
severity. Statistically, confirmed COVID-19 cases worldwide are 100
times higher than the confirmed cases of SARS and MERS. This is
because, 1) SARS-CoV-2 replicates to much higher levels in the nose
and mouth than SARS and MERS, and 2) this leads to very high levels
of virus shedding in the environment by people who are either
pre-symptomatic or asymptomatic. Thus, a huge percentage of
infected people can transmit the virus without realizing that they
are even infected.
[0007] Rapid detection methods independent of lab setting have been
identified as one of the foremost priorities for promoting epidemic
prevention and control. Currently, there are two main strategies
for the detection of COVID-19. The first is a real time reverse
transcriptase (RT) polymerase chain reaction (RT PCR)-based
strategy that detects the viral nucleic acid in patient samples
(presence of the viral RNA). The second strategy is an
immunological assay that detects viral protein antigens or serum
antibodies produced as a result of the body's immune response to
the viral infection. The two strategies complement each other, with
the qPCR strategy detecting the virus during its active phase,
while the immunological assay identifies individuals who have
developed antibodies to fight the disease. An interesting approach
to COVID-19 diagnosis that utilizes either of these strategies, is
by the use of biosensors (12, 13). Biosensors interact with
biomolecules and transduce their readings into measurable outputs,
such as optical, electrical and enzymatic. Biosensors can provide
label-free, real-time detection and curtailment of non-specific
binding. However, they also face the challenge of efficient
immobilization of biomolecules on the sensing surface (14).
[0008] Currently, the molecular technique of quantitative real time
polymerase chain reaction (qRT PCR) is the gold standard for
SARS-CoV-2 detection using samples from respiratory secretions.
However, it is a time consuming and cumbersome procedure that takes
long processing times over days for results. Several other
molecular assays have been developed to detect SARS-CoV-2, such as
enzyme-based assays like ELISAs, and rapid tests that aim to detect
either antibodies against the virus or the viral antigen
themselves. Nevertheless, most of these antigen-antibody-based
assays have failed quality control due to their rapid development
without proper testing and result in either false negative or false
positive detection due to the long time it takes to develop serum
responses to the viral infection (from days to two weeks).
[0009] Recent reports have found distinctive UV-visible light
spectra in the range of 250-800 nm for proteins rich in charged
amino acids, are in monomeric form, and devoid of aromatic amino
acids (15-19). Prasad et al. have shown that the protein charge
transfer spectra (ProCharTS) band comprises of facile photoinduced
peptide backbone to side chain charge transfer and side chain to
side chain charge transfer transitions in charged amino acids such
as lysine (Lys) and glutamate (Glu) (18). In fact, all naturally
occurring charged amino acids can be identified as either electron
donor (D), bridge (B), or electron acceptor (A) units. Within
protein folds, the charged side chains of these amino acids (e.g.,
Lys amino or Glu carboxyl groups), and the peptide backbone play
the role of D and A groups, while the aliphatic (non-polar) part of
the side chain or the intervening protein/solvent medium forms the
B component of the D-B-A units. While CT transitions for charged
amino acid monomers are expected to be in the deep UV (below 250
nm), strong inter-residue electronic couplings imposed by protein
folds can shift such transitions dramatically to the visible end.
Previous molecular dynamics (MD) and electronic structure analysis
revealed three specific factors which primarily determine the
ProCharTS absorption range: (1) distance between the side chains of
charged amino acids (which could be in the range of 2-10 A or
more), (2) the charge complementarity of the interacting side
chains and (3) the medium pH (19). Solvation and conformations of
the amino acid side chains and the peptide backbone were also shown
to modulate the spectral range of the CT transitions to a lesser
extent (18).
[0010] Electrostatic analysis has been used in related art to study
protein interactions at a structural level. For example, it was
observed that the protein surface of ACE2 shows negative
electrostatic potential, while the S proteins of the
SARS-CoV/SARS-CoV-2 exhibit positive potential (20). Additionally,
it was found that SARS-CoV-2 S protein is slightly more positively
charged than that of SARS-CoV, giving it a higher affinity to bind
to negatively charged regions of the ACE2 (30% higher binding
energy (7)). Similarly, the SARS-CoV-2 N protein has three distinct
but highly conserved parts: the N-terminal RNA-binding domain (NTD)
which is responsible for RNA binding via its distinct basic
(positively charged) finger and palm regions: a C-terminal
dimerization domain (CTD) which is responsible for oligomerization,
and intrinsically disordered central Ser/Arg (SR)-rich linker which
is responsible for linker for primary phosphorylation, respectively
(21).
[0011] Thus, most of the methods used so far either require skilled
manpower and are time consuming if accurate, or not reliable at
all, if fast. On the other hand, biosensor technology provides
excellent sensitivity, but requires metal coating deposited on the
device, thereby raising cost. Furthermore, some of these biosensors
suffer from temperature-dependence which can be a hindrance for
portable biosensors in outdoor conditions. Some require expensive
reagents and reaction times are often longer.
RELATED ART
[0012] Examples of related art with different assay methods and
analysing techniques are found in various publications. For
example, the patent publication US2017362668A1 to Meso Scale
Technologies with the title Co-binder assisted assay methods
disclose methods for reducing cross-reactivity between species
employed in multiplexed immunoassays.
[0013] Another example of related art is found in the patent
publication US2021123883A1 to University of Utah Research
Foundation with the title Whole virus quantum mechanical tunneling
current and electronic sensors. This publication discloses a field
effect transistor (FET) biosensor for virus detection of a selected
virus within a sample volume.
[0014] A further example of related is the patent publication
WO2021081476A1 to University of Utah Research Foundation with the
title Zero Power visible colorimetric pathogen sensors. This piece
of related art shows a method in which a visibly perceived
colorimetric pathogen sensor comprises a substrate and a molecular
recognition group coupled to the substrate. The molecular
recognition group can bind a target pathogen and when that occurs,
the reflected light cab be altered thereby changing apparent color,
thus indicating the detected target pathogen.
[0015] The article with the title An Analysis Review of Detection
Coronavirus Disease 2019 (COVID-19) Based on Biosensor Application
by Bakr Ahmed Taha et al. found on the web address
https://www.mdpi.com/1424-8220/20/23/6764/summarizes technologies
for the detection of coronavirus disease 2019 (COVID-19)
technologies with biosensors that operate using laser detection
technology.
[0016] The related art patent publication WO2011060184 to Cermed
Corporation with the title Cervical cancer screening by molecular
detection of human papillomavirus-induced neoplasia further shows
point-of-care tools for screening biological samples for markers
associated with pathogenic microbial infections. This publication
discloses a technology for screening cervical cells for the
expression of proteins that occur because of human papillomavirus
infection and progression to invasive cervical cancer.
[0017] Another related art patent publication WO2015116083 to
Hewlett Packard Development with the title Microfluidic sensing
device. This publication discloses a microfluidic sensing device
that comprises a channel and an impedance sensor within the
channel. A particle in a fluid passing the sensor is identified
based on the sensed impedance characteristics.
[0018] There is a need in the field for fast, cheap and accurate
methods of detection of a virus such as SARS-CoV-2, which may be
used to slow the spread of the virus till a vaccine or effective
therapy can be found.
SUMMARY OF THE INVENTION
[0019] The present disclosure describes embodiments for a method
and system for virus detection and monitoring.
[0020] The current disclosure describes embodiments from several
aspects. Some aspects describe a system and method to detect and
monitor the possible binding or interactions between proteins and
proteins, proteins and antibody, viruses and proteins, viruses and
antibodies, cells and proteins, cells and antibodies as well as
virus-cell, or cell-cell interactions, or any interactions between
any part of protein, antibody, virus, cell with any part of
protein, antibody, virus, cell of the same or different kind.
Embodiments are based on and utilize the higher positive
electrostatic potential of SARS-CoV-2 N protein and its antibody
for an opto-electrical detection method and system to successfully
detect virus infection in nasal swabs from SARS-CoV-2-infected
individuals.
[0021] System embodiments incorporate a source capable of emitting
light, natural or manmade. The emitted light spectrum can cover the
whole spectrum. This light passes through a sample, and its
absorbance, reflectance, transmittance or any other type of
scattering, corresponding intensities or any other possible forms
that could be extracted out of these intensities and can be
collected using a spectrometer or any light-based detector, sensor
or device. These intensities are measured and recorded over a
time-period or can be mapped to the time domain in order to
determine, measure or study light scattering parameters.
[0022] Embodiments comprise a transparent container allowing light
to pass through the material without appreciable scattering of
light configured for holding a sample. The sample under test can be
loaded into the transparent container, which in different variants
is any type of plate, box, tube, or any type of paper made out of
transparent material or any other material, allowing light to pass
through the material without appreciable scattering of light. The
sample or specimen can either be tested in place or can be taken
out to be tested away from its origin.
[0023] The light-based source, the light-based detector and the
sample is in different embodiments aligned with each other for
example in a straight line (i.e., source-sample-detector) or any
other possible alignment that enables the measurement of the
mentioned intensities.
[0024] During the measurement, the sample can be added to the
appropriate container, applying the measurement conditions,
collecting the responses, either directly measured intensities are
collected and/or direct relationship with time is established or
the direct measured data are further processed to extract
parameters or set of parameters and correlate them with time to
establish the relationship.
[0025] This relationship is further considered to determine the
type of interaction either by direct judgment or further
processing. A mathematical relation can be constructed to represent
the interaction process. For example, if within the time-period,
this relationship shows nonlinearity, then this means that the
interaction took place; if it shows a constant behaviour, then the
interaction did not take place. In case of linear relationship that
can be represented with a slope, the two kinds of elements or more
than two elements under investigation, their individual intensities
should be measured and considered accordingly.
[0026] The proposed method can monitor vaccine or drug development
process.
[0027] Different embodiments comprise any type of equipment that
can measure light intensity is also included. For example, a camera
or any possible image-based system that can take either video or
pictures or that has the ability to map light intensities to any
possible form, or its output can be processed further. Embodiments
are configured to utilize the phenomenon that when two proteins
bind or a molecular interaction takes place between them, due to
this interaction, a possible form of kinetic energy is produced,
for example as a cloud. This kinetic energy can cause disturbances,
such as altering the Brownian motion of the molecules that can
change or alter light distribution on the surface of the sample or
inside. The generation of a cloud-like response due to the
biochemical interaction would also disturb the light intensity
distribution and accordingly the measured light intensities.
[0028] Further embodiments use techniques and equipment for
conducting chronoamperometry or chronopotentiometry measurements or
other measurements based on this principle to detect and monitor
such interactions/binding. The measured value, either current or
voltage over time, are used in different embodiments to explore the
interaction. When an interaction occurs, the measured profile over
time will increase or decrease in trend, depending upon the nature
of the test samples, resulting in either an exponential growth or a
decay behaviour. Furthermore, in other embodiments current voltage
or capacitance voltage, impedance, or any other possible
electrical-based measurements are used for this purpose.
[0029] Low and high frequency, optical and/or electrical scattering
parameters or propagating waves, or any other possible form of
measurements are used in yet other embodiments.
[0030] The linear electro-optic effect is the change in the index
of refraction that is proportional to the magnitude of an
externally applied electric field. The electro-optic effect may
also be non-linear, for example over a wider range. Hence, the
measured light intensity varies with DC electric voltage. The DC
bias will also change the current and capacitance as they depend on
the applied voltage. Values for the capacitance can for example be
extracted from scattering parameters or impedance measurements or
from determined relations between the parameters. This
relationship/dependency can be converted to a chart or be expressed
as a relationship that could be used for detection enhancement and
further identification of the sample under test. For a specific
virus type, the corresponding capacitance and light intensity at
zero bias could also be used for further detection purposes. In
embodiments, the voltage dependency is used to provide more
information, for example about the viral load and to identify the
viral stage infection.
[0031] In embodiments sample strips are provided to collect a
sample of analytes from a test object such as a patient, a fluid or
a physical object, or to store a sample of analytes collected from
such test objects e.g., for later analysis. Sample strips are in
embodiments configured with a portion coated with antibodies. Such
sample strips coated with antibodies are preferably designed as
flexible strips and are preferably kept inside packs with probe
medium for granting their lifetime and functionality. These sample
strips are in embodiments configured to be used directly loading a
sample from a nasal swab or some other probe. In other embodiments
the sample strip is configured to be formed and attached to a probe
to be inserted inside the nose. In embodiments, such sample strips
may also be coated with a probe electrode to conduct current
measurements.
[0032] Embodiments comprises a device for nasal sampling configured
to be insertable inside the nose of a human test object. Some of
these embodiments are configured such that a sample strip is
attachable at the end of an elongate section of the device. The
sample strip is attached to touch the nasal walls when the nasal
sampling device is inserted into the nose. Embodiments of this
sampling device is probed with or comprises two electrodes
configured to apply current over time continuously through a chip
integrated within it, such that an electric field is applied over a
sample of analytes collected on the sample strip. In embodiments,
an integrated light source and detector are incorporated in the
sampling device and configured to expose the sample to light and to
detect light passing through the sample, enabling the capability to
conduct light intensity measurements non-invasively.
[0033] The concept of strips can be further used to detect
different samples or specimens taken from blood, breath, urine,
nasal swabs, stool, etc. For example, the subject can breathe,
exhale, or sniff into a device such as embodiments configured with
a probe coated with antibodies. If binding occurs, the integrated
device should be able to pick up the interaction, confirming that
the patient is infected with the virus to which the antibodies are
directed against.
[0034] An example of embodiments for the use of the technologies
specified herein is provided for SARS-CoV-2 diagnosis, though
depending upon the antibody used, it is in other embodiments
configured for the detection of any respiratory virus such as
influenza, respiratory syncytial virus (RSV), adenoviruses, or
other coronaviruses like SARS and MERS. The antibody against the
virus in question is in embodiments be coated on flexible sample
strips for increasing their lifetime and functionality and kept
inside packs with an Opto-electric probe. The antibody coated
sample strips are in embodiments configured to be used either
directly for loading the nasal swab sample or as in other
embodiments configured to be attached directly to the probe to be
inserted inside the nose. In embodiments, the sample strips are
coated with an electrode and be configured to conduct current
measurements directly, in which case they serve as the probe
themselves. This embodiment of probe is configured to be inserted
inside the nose, allowing it to touch the nasal wall. In
embodiments, the probe is configured to apply current over time
continuously through a chip integrated within it, thereby applying
an electric field over a sample. Also, an integrated light source
and detector is in embodiments incorporated at the other end to
conduct light intensity measurements non-invasively.
[0035] The concept of strips can be further used to detect
different specimens taken from blood, breath, urine, nasal swabs,
stool, etc. For example, the test subject can breathe, exhale, or
sniff into a device with a probe coated with antibodies, in
accordance with embodiments. If binding or interaction with
antibodies on the sample strip occurs, embodiments with the
integrated device as described above is configured to be able to
pick up the interaction, confirming that the patient is infected
with the virus to which the antibodies are directed against.
[0036] Method and system embodiments are configured such that: a
said sample is placed and distributed in one or more microfluidic
channels; measurement of said light scattering parameters and/or
said electrical parameters is conducted for the sample content in
each of said microfluidic channels; and virus concentration and/or
virus load is determined based on said measured parameters.
[0037] In further variants of such method and system embodiments
said sample is diluted such that there is stepwise increasing
dilution of antibody content in said one or more microfluidic
channels and said measurement of parameters is conducted for said
dilution steps.
[0038] In method and system embodiments said sample is placed and
distributed in a plurality of parallel microfluidic channels; the
antibody content is serially diluted in said plurality of parallel
channels, simultaneously measuring said parameters in said
plurality of channels; and said virus concentration and/or virus
load is determined based on said measured parameters.
[0039] Method and system embodiments are configured such that the
virus concentration and/or virus load is based on a mathematical
relationship between said parameters and virus concentration and/or
virus load, said mathematical relationship for example being
calibrated against known virus concentration or virus load.
[0040] Other aspects and embodiments of the invention will be
apparent as will be shown in the detailed description of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0042] The accompanying drawings illustrate non-limiting example
embodiments of the invention.
[0043] FIG. 1 shows an exemplifying illustration and modelling of
binding mechanisms, wherein it is shown in: (a) SARS-CoV-2 binding
with host cell illustration. (b) Binding of ACE2 and Spike protein
along with illustration of the spike protein subunits, S1 and S2.
(c) Schematically showing the distribution of the spike protein in
suspension. (d) As an example, schematically showing the
distribution of ACE2 in suspension. (e) As an example,
schematically showing the distribution of ACE2 and S protein after
binding.
[0044] FIG. 2 shows an illustration of the concept of optical
detection in accordance with embodiments in an experimental design
setup, wherein: FIG. 2(a) shows an embodiment of an optical
measurement setup consisting of a smart phone as a light source and
a mini-spectrometer utilized to collect light waves passing through
the sample kept in a holder. FIG. 2(b) shows a graph of the smart
phone power spectra versus wavelength. FIG. 2(c) shows an
illustration of the spectrometer detection principle.
[0045] FIG. 3 shows graphs of an example of optical responses for
spike proteins S1 and S2 along with their corresponding blanks,
wherein:
[0046] FIG. 3(a) shows measured responses for spikes proteins S1
and S2 at the highest concentration individually (S1B and S2B,
respectively), along with their corresponding blanks.
[0047] FIG. 3(b) shows time domain measurements of a
microcentrifuge tube.
[0048] FIG. 3(c) shows measured optical responses for the mixed
proteins versus time.
[0049] FIG. 3(d) shows relative change in light intensity per light
path versus loaded mass.
[0050] FIG. 4 shows an illustration of light intensity and its path
length, wherein:
FIG. 4(a) shows a blank representation, and FIG. 4(b) shows the
light path length of a sample. L and A are the length and
cross-sectional area of container, l is the light path length.
I.sub.0 is the incident light intensity, I.sub.b is the intensity
of light passing through the blank and I instantaneous is the light
intensity passing through the sample. FIG. 4(c) shows loaded mass
versus relative change in light intensity per light-path length.
The measured points have been fitted with exponential function
expressed by equation (1) below with the following parameters:
m.sub.i=1.003.mu..+-.2.68n, m.sub.f=2.163.mu..+-.34.7n and
.alpha.-factor is 1.28435.+-.0.030. The other fitting model
accuracy parameters are: Reduced Chi-Sqr, R-Square (COD), Adj.
R-Square are 28.8 atto, 1 and 1, respectively, which indicates the
best possible fit.
[0051] FIG. 5 shows graphs of optical detection of binding
interactions, in this example between ACE2 and other proteins,
wherein:
[0052] FIG. 5(a) shows measured light intensities over time for
individual assessment of ACE2, S1A(S1X), S1B(S1Y), and BSA.
[0053] FIG. 5(b) shows the measured mixed light intensities versus
time for ACE2 mixed with either S1A(S1X), or S1B(S1Y), or BSA.
[0054] FIG. 5(c) shows the measured ACE2-S1A interaction profile
for an extended time period.
[0055] FIG. 5(d) shows extracted slopes for the individual and
mixed protein suspensions.
[0056] FIG. 6 shows graphs of optical detection of the binding
affinities between proteins and antibodies, wherein:
[0057] FIG. 6 (a) shows the receptor binding domain (RBD) of the
spike protein with its antibody (AB).
[0058] FIG. 6(b) shows the nucleocapsid protein (NCP) and its
antibody.
[0059] FIG. 6(c) shows NCP binding with the antibody after mixing
inside.
[0060] FIG. 6(d) shows NCP binding with the antibody after mixing
outside.
[0061] FIG. 7 shows graphs of opto-electrical measurements of
nucleocapsid protein (indicated NCP or NC protein) with direct
current DC biasing, wherein:
[0062] FIG. 7(a) shows measured NC protein optical response versus
time at different DC bias voltages.
[0063] FIG. 7(b) shows binding measurements between NC protein and
its corresponding antibody after subjected the solution to an
electric field.
[0064] FIG. 8 shows graphs of illustrating examples of
protein-protein interaction measurements on paper based
nitrocellulose membrane (NM), wherein:
[0065] FIG. 8(a) shows optical responses on nitrocellulose membrane
(NM) alone, nitrocellulose membrane and spike protein (NM+P), and
nitrocellulose membrane and antibody to spike protein (NM+AB)
alone.
[0066] FIG. 8(b) shows optical responses to spike protein-antibody
binding on the nitrocellulose membrane.
[0067] FIG. 9 shows a graph of chronoamperometry measurements
indicating current versus time for different sample suspensions, in
accordance with embodiments.
DETAILED DESCRIPTION
[0068] Throughout the following description, specific details are
set forth in order to provide a more thorough understanding to
persons skilled in the art. However, well known elements may not
have been shown or described in detail to avoid unnecessarily
obscuring the disclosure of embodiments. The following description
of examples of the technology is not intended to be exhaustive or
to limit the system to the precise forms of any example embodiment.
Accordingly, the description and drawings are to be regarded in an
illustrative, rather than a restrictive, sense.
[0069] Optical, label-free biosensors have been utilized frequently
in biomolecular detection due to their continuous monitoring and
high sensitivity to local variation, including the refractive index
change. They are capable of detecting interactions between
molecules and their surrounding media.
[0070] On a general level, embodiments comprise a system and a
method to detect and possibly monitor the possible binding or
interactions between proteins and proteins, proteins and antibody,
viruses and proteins, viruses and antibodies, cells and proteins,
cells and antibodies. Also, virus-cell, or cell-cell interactions,
or any interactions between any part of protein, antibody, virus,
cell with any part of protein, antibody, virus, cell of the same or
different kind.
[0071] Embodiments comprises a method of opto-electrical detection
of the presence of possible bindings or interactions between
analytes in a sample, comprising: exposing a sample to light from a
light source; detecting light passing through the sample; applying
an electrical field over the sample; determining the values of a
selection of light scattering parameters for the light passing
through the sample in response to the electrical field; and/or
[0072] determining the values of a selection of electrical
parameters, such as electrical scattering parameters, in response
to the electrical field; determining the presence of bindings or
interactions between analytes in the sample based on the values of
the determined light scattering parameters and/or the determined
values of the electrical parameters, for example electrical
scattering parameters.
[0073] Further embodiments comprises a system of opto-electrical
detection of the presence of possible bindings or interactions
between analytes in a sample, comprising: a light source configured
to emit or transfer natural or manmade light and to expose a sample
with said light; an electric field device configured to apply a
biasing electric field over the sample; a light detector configured
to detect light passing through a said light exposed sample; an
electric parameter detector configured to detect electric
parameters. Such embodiments further comprise a processing device
having code portions configured direct the processor to: determine
the values of a selection of one or more light scattering
parameters of the detected light passing through a said light
exposed sample; determine the values of a selection of electrical
parameters, for example electrical scattering parameters, in
response to the electrical field, and to determine the presence of
bindings or interactions between analytes in the sample based on
the values of the determined light scattering parameters and the
determined values of the electrical parameters, for example
electrical scattering parameters.
[0074] An underlying mechanism for the concept of embodiments is
that when two proteins bind or a molecular interaction takes place
between them, due to this interaction, a possible form of kinetic
energy is produced, perhaps emitted as a cloud. This kinetic energy
can cause disturbances, such as altering the Brownian motion of the
molecules which can change or alter light distribution on the
surface of the sample or inside the sample. The generation of a
cloud-like response due to the biochemical interaction should also
disturb the light intensity distribution and accordingly the
measured light intensities.
[0075] Further, the linear electro-optic effect is the change in
the index of refraction that is proportional to the magnitude of an
externally applied electric field. As mentioned herein. the
electro-optic effect may also be non-linear, for example over a
wider range. Hence, the measured light intensity varies with DC
electric voltage. The DC bias will also change the current and
capacitance as they depend on the applied voltage. This
relationship/dependency may for example be converted to a chart
that could be used for detection enhancement and further
identification of the sample under test. For a specific virus type,
the corresponding capacitance and light intensity at zero bias
could also be used for further detection purposes.
[0076] In method embodiments, the presence of bindings or
interactions between analytes in the sample is determined is
determined based on a determined characteristic of the detected
light scattering parameters, and/or electrical scattering
parameters for specific values of the electric parameters. In
system embodiments, the processing device comprises code portions
configured to determine the presence of bindings or interactions in
the sample based on determined characteristic of the detected light
scattering parameters, and/or electrical scattering parameters, for
specific values of the electric parameters.
[0077] Method embodiments comprises determining the presence of
bindings or interactions between analytes in the sample by
comparing the discrepancy between the measured responses at
different applied electrical fields. In system embodiments, for
this purpose the processing device comprises code portions
configured to determine the presence of bindings or interactions
between analytes in the sample by comparing the discrepancy between
the measured responses at different applied electrical fields.
Further method and system embodiments are configured to determine
the presence of bindings or interactions between analytes in the
sample by comparing the discrepancy between the measured responses
at different applied electrical fields; and/or wherein the measured
light intensity varies with DC electric voltage and/or values for
the capacitance, for example, can be extracted from scattering
parameters or impedance measurements or from determined relations
between the parameters; and/or to determine information about the
viral load and/or identifying the viral stage infection based on
the voltage dependency. In embodiments of system, the processing
device comprises code portions configured account for these
possible features.
[0078] The emitted or transferred, manmade or natural, light from
the light source may comprise the whole spectrum of
wavelengths.
[0079] In such method and system embodiments the light detector is
one or more of: an image-based system such as a camera, a
spectrometer or any light-based detector, sensor or device that is
capable to detect light intensity dependent or derivable parameter
values. The method and system are in embodiments configured to
detect light intensity and to determine light scattering parameters
based on one or more of absorbance, reflectance, transmittance or
any other type of light scattering, corresponding intensity or
other parameter that is extractable or collectable by means of a
light detector.
[0080] In method and system embodiments a selection of one or more
light scattering parameters, for example intensities, are measured
and possibly recorded over a time-period and/or mapped to a time
domain. In system embodiments the processing device comprises code
portions configured to measure and possibly record a selection of
one or more light scattering parameters over a time-period and/or
to map said parameters to a time domain.
[0081] In method and system embodiments the sample is loaded into a
transparent holder or container allowing light to pass through the
material without appreciable scattering of light and being
configured for holding a sample, for example on or more of a plate,
box, tube or any type of transparent paper or other transparent
material. For this purpose, system embodiments, may further
comprise a transparent holder or container allowing light to pass
through the material without appreciable scattering of light and
being configured for holding a sample, for example on or more of a
plate, box, tube or any type of transparent paper or other
transparent material.
[0082] A sample may, as in method embodiments, be tested in place
in close connection with or in the proximity of the taking of the
sample or be stored and/or transported for testing spatially and/or
temporally remote from the taking of the sample. For this purpose,
in system embodiments, the transparent holder or container is
configured to store and/or transport a sample.
[0083] In method and system embodiments, the light source, a sample
and the light detector are aligned such that measurement of light
passing through the sample is enabled, for example by alignment in
a straight line or other possible such alignment.
[0084] The electric field device comprises, in method and system
embodiments, two electrodes configured or configurable at
respective sides of a sample and being coupled or couplable to an
electric energy source.
[0085] In method and system embodiments, the sample is added to a
holder or container aligned with the light source and the light
detector such that measurement of light passing through the sample
is enabled; and measured responses in the form of the determined
values of said selection of optical or electrical parameters, for
example electrical scattering parameters, in response to the
electrical field are collected to determine the presence of
bindings or interactions between analytes in the sample. In system
embodiments, the processing device comprises code portions
configured to collect measured responses in the form of the
determined values of said selection of optical or electrical
parameters, for example electrical scattering parameters, in
response to the electrical field for the sample that is added to a
holder or container aligned with the light source and the light
detector such that measurement of light passing through the sample
is enabled.
[0086] Such method embodiments comprises steps, and in such system
embodiments the processing device comprises code portions,
configured to collect measured responses comprising one or more of:
collecting directly measured light intensity; and/or collecting
directly measured light intensity and establishing direct
relationship with time; and/or processing direct measured data to
extract parameters or a set of parameters and correlating said
parameters with time to establish relationship with the presence of
bindings or interactions between analytes in the sample.
[0087] Method and system embodiments further comprises determining
the type of binding and/or interaction between analytes in the
sample based on a relationship between the values of determined
light scattering parameters and/or of determined electrical
scattering parameters, and time, for example by direct judgement or
by further processing. For this purpose, in system embodiments the
processing device comprises code portions configured to determine
the type of binding and/or interaction between analytes in the
sample based on a relationship between the values of the determined
light scattering parameters and time, and/or of the electrical
scattering parameters and time.
[0088] In method and system embodiments, a mathematical relation
for the determined parameters represents the process of bindings or
interactions between analytes in the sample. For this purpose, in
system embodiments, the processing device comprises code portions
configured to apply a mathematical relation for the determined
parameters to represent the process of bindings or interactions
between analytes in the sample.
[0089] In examples of such method and system embodiments, with a
mathematical relation between determined light scattering
parameters, and/or electrical scattering parameters, and time for
measured responses at selected applied electrical fields, binding
and/or interaction between analytes is determined to be present if
the relations shows nonlinearity and not to be present if the
relation shows constant behaviour. In system embodiments, for this
purpose, the processing device comprises code portions configured
to, for a mathematical relation between determined light scattering
parameters, and/or electrical scattering parameters, and time for
measured responses at selected applied electrical fields, determine
that binding and/or interaction between analytes is present if the
relations shows non nonlinearity and not present if the relation
shows constant behaviour. In case of linear relationship that can
be represented with a slope, the two kind of elements or more than
two elements under investigation, their individual intensities
should be measured and considered accordingly, in method and
corresponding system embodiments. Further, in such embodiments the
non-linearity may be expressed in terms of extracted single or
multi-parameters, the non-linearity may be observed manually or
automatically and the variation of response profiles of the one or
more parameters may be detected over time. In corresponding system
embodiments, the processing device comprises code portions
configured to express the non-linearity in terms of extracted
single or multi-parameters, to enable the non-linearity to be
observed manually or automatically and to detect the variation of
response profiles of the one or more parameters over time. Further
method embodiments and corresponding system embodiments comprises
are configured to determining a mathematical relation for the light
scattering parameters, and/or electrical scattering parameters, and
values of the electric parameters to represent the presence and/or
the process of one or more of said bindings or interactions between
analytes in the sample based on the value of light scattering
parameters for the detected light passing through the sample.
[0090] Method and system embodiments further comprise the use of
and configuration with a chronoamperometry or a chronopotentiometry
measurement method used to detect and/or monitor the presence
and/or the process of one or more of said bindings or
interactions.
[0091] In method and system embodiments the measured value, for
example either current or voltage over time, can be used to explore
the interaction. When an interaction occurs, the measured profile
over time will increase or decrease in trend, depending upon the
nature of the test samples, resulting in either an exponential
growth or a decay behaviour. Furthermore, current voltage or
capacitance voltage, impedance, or any other possible
electrical-based measurements can also be considered. In method
embodiments, the presence and/or the process of one or more of said
bindings or interactions is determined, detected and/or monitored
based on measurements of an electrical parameter, for example an
electrical scattering parameter, for example a parameter based on
one or more of the voltage, the current, the capacitance and/or the
impedance over time in relation to a said applied electric field.
For this purpose, in system embodiments, the processing device
comprises code portions configured to determine, detect and/or
monitor the presence and/or the process of one or more of said
bindings or interactions based on measurements of an electrical
parameter, for example one or more of the voltage, the current, the
capacitance and/or the impedance over time in relation to a said
applied electric field.
[0092] In further embodiments of method and system, low and high
frequency scattering parameters or propagating waves, or any other
possible form of measurements can also be considered. For this
purpose, method and system embodiments, further comprise or are
configured to measure and process parameter values based on low and
high frequency scattering parameters or propagating waves.
[0093] In method embodiments, and in corresponding system
embodiments comprising configured code portions, a characteristic
of the determined light scattering parameters, and/or electrical
scattering parameters, for specific values of the electric
parameters is determined to represent the response of the
determined light scattering parameters and/or electrical scattering
parameters, due to the possible binding or interaction between one
or more analytes in a sample, such as between proteins and
proteins, proteins and antibody, viruses and proteins, viruses and
antibodies, cells and proteins, cells and antibodies as well as
virus-cell, or cell-cell interactions, or any interactions between
any part of protein, antibody, virus, cell with any part of
protein, antibody, virus, cell of the same or different kind.
[0094] Method and system embodiments are configured and/or used to
monitor a vaccine or drug development process.
[0095] Method and system embodiments described herein may be
configured for virus detection. A method embodiment is for this
purpose used for virus detection and comprises collecting and
processing electrical and optical responses individually or
simultaneously to extract a set of parameters for detection,
quantification and identification of virus. In a system embodiment
for this purpose, the processing device comprises code portions
configured to collecting and processing electrical and optical
responses individually or simultaneously to extract a set of
parameters for detection, quantification and identification of
virus. In variants, such method and system embodiments comprise, or
are configured to enable that, a virus cell is contacted by a
selected antibody in a sample. Variants of such method and system
embodiments are further adapted and used for detection of one or
more of virus from the group SARS-CoV-2, SARS, MERS, influenza,
respiratory syncytial virus (RSV), adenoviruses, or any other
respiratory virus.
[0096] Further embodiments of the method and the system adapted for
virus detection, further comprises or are configured to applying an
electrical pulse over or through a sample containing analytes from
a test subject and selected antibodies, thereby enabling
electro-insertion of said antibodies into any virus cell present in
the sample.
[0097] In method and system embodiments adapted for virus
detection, a virus cell in a sample is suspended in an aqueous
transport medium of a nasopharyngeal swab along with anti-N
antibodies (anti-N).
[0098] In further method and system embodiment adapted for virus
detection, the method comprises steps, and the system the
processing device comprises code portions, configured to determine
a characteristic of the light scattering parameters, and/or
electrical scattering parameters, for specific values of the
electric parameters and to measure the viral nucleocapsid protein
and anti-N antibody interactions in a sample to differentiate
between SARS-CoV-2 negative and positive nasal swab samples.
[0099] Embodiments of the method and the system adapted for virus
detection, further comprises a sample strip configured for
collecting a sample of analytes from a test object, the sample
strip comprising a portion coated with antibodies. In variants, the
sample strip is coated with an antibody configured to bind or
interact with of one or more of virus from the group SARS-CoV-2,
SARS, MERS, influenza, respiratory syncytial virus (RSV),
adenoviruses, or other respiratory virus.
[0100] In further method and system embodiments adapted for virus
detection, the sample strip comprises an electrode coated thereon
and configured to conduct current measurements directly.
[0101] Other method and system embodiments for opto-electric virus
detection, further comprises a device for nasal sampling configured
such that a sampling strip is attachable at a section of the nasal
sampling device that is configured to be insertable in the nose of
a human test object, the nasal sampling device comprising two
electrodes configured to apply an electric field over a sample of
analytes collected on a said sampling strip. In such system
embodiments the nasal sampling device may further comprise an
integrated light source and detector configured to expose the
sample to light and to detect light passing through the sample.
[0102] Such integrated method and system embodiments are, in
variants, configured such that when the presence of a binding or
interaction between a virus and an antibody is detected in a sample
from a patient, an indication signal is presented confirming that
patient is infected with the tested virus. The indication signal
for infection may for example be in the form of light, sound, text
or image presented via a corresponding presentation device.
[0103] In embodiments of method and system, a sample is taken from
bodily tissue or fluid, for example blood, breath, urine, nasal
swabs, stool, in the system embodiments with a strip configured for
this purpose.
[0104] These method and system embodiments are further described
and explained by illustrating examples below.
[0105] FIG. 1 shows an exemplifying illustration and modelling of
binding mechanisms of virus to surrounding media. FIG. 1(a)
schematically illustrates SARS-CoV-2 binding with a host cell. FIG.
1(b) In this example, binding of ACE2 (angiotensin converting
enzyme 2) and Spike protein along with illustration of the spike
protein subunits, S1 and S2. FIG. 1(c) schematically shows the
distribution of the, in this example, spike protein in suspension.
FIG. 1(d) schematically shows the distribution of ACE2 in
suspension. FIG. 1(e) In this example, schematically shows the
distribution of ACE2 and S protein after binding in suspension.
[0106] There are several antigens and elements that in SARS-CoV2
virus that are active in binding and/or interaction, ACE2 is one
example, nucleocapsid is another example and the technology of the
present disclosure is applicable to other antigens and elements as
well. The technology of embodiments disclosed herein can, as
described herein, be applied on any virus, antigens, antibodies or
elements.
[0107] For the sake of example, embodiments herein are explained
with application of the technology on ACE2 and nucleocapsid. In
terms of detection, one of the most prominent features of the
SARS-CoV-2 virus, like other coronaviruses, is the spike protein
(S) that protrudes out of the virus particle essentially like
"spikes"; hence the name. The spike protein forms a trimer that is
used by the virus to enter susceptible cells using the
angiotensin-converting enzyme 2 (ACE2) protein as the cellular
receptor, the same protein used by the SARS-CoV-1 virus that caused
the first SARS epidemic in 2003 (FIG. 1(a)). The spike protein is
cleaved by host cell membrane proteases into two subunits: the
surface subunit S1, and the transmembrane subunit S2 (FIG. 1(b). It
is the surface S1 subunit that is used by the virus to interact
with ACE2 protein using its receptor binding domain (RBD). This
allows the virus to attach to the susceptible cells, while the S2
protein is used for the actual fusion of the virus with the cell
membrane, allowing the virus to be endocytosed into the cytoplasm
and release its genomic RNA cargo, wrapped up in the nucleocapsid
protein (NCP), into the cytoplasm. It is this RNA that is
immediately used to translate viral proteins and use them for
successful virus replication in the susceptible cells. The spike
protein is also the most immunogenic domain of the virus towards
which most of the neutralizing antibody responses against the virus
are generated in infected individuals, making it an ideal candidate
for vaccine as well as a target of drug development. In the context
of COVID-19, it has been observed that the SARS-CoV-2 spike
glycoprotein binds ACE2 with much higher affinity than SARS-CoV-1
spike protein, which may explain the higher transmissibility of
SARS-CoV-2 in the human population.
[0108] Embodiments described herein, comprises a light-based method
to detect SARS-CoV-2 and potentially disrupt its binding ability
with its receptor, rendering the virus non-infectious by combining
optical detection with electric current. The measured intensity of
light is used to determine information about different cellular
parameters in a sample under study with method embodiments. Light
scattering and electrical scattering are correlated with cell/virus
size and reflect the complexity of their exterior or interior
structures. When light passes through a cell, the intensity is
associated with the DNA/RNA content of the cells. The nuclei size,
cell shape and the refractive index variation of cells contribute
to light intensity in the cell. It is worth adding that the
measured optical spectrum of light passing through a sample
consists of many features that in embodiments is used to reveal and
determine important information about the sample under test.
Usually when the cell/tissue changes from a normal state to
infectious, metabolic alterations and genetic modifications occur.
This causes dramatic changes in their physiological, biochemical
and morphological characteristics. Indeed, the metabolic
differences between infected and normal cells leads to several
variations in cellular parameters, such as their size. In this
text, embodiments with an optical label-free detection method
incorporating a smartphone light source and a portable mini
spectrometer for SARS-CoV-2 detection are described. The detection
of parameters reflecting light interactions with control and viral
protein solutions in accordance with embodiments enables a quick
decision regarding whether a sample under test is positive or
negative, thus enabling SARS-CoV-2 detection in a rapid and
label-free manner.
[0109] FIG. 2 illustrates a concept of optical detection in
accordance with embodiments in an experimental design setup. FIG.
2(b) shows a graph of the smart phone power spectra versus
wavelength and FIG. 2(c) shows an illustration of the spectrometer
detection principle.
[0110] An experimental setup utilized to illustrate an exemplary
embodiment presented in FIG. 2(a), incorporating a mini
spectrometer and a smart mobile phone that was employed as a light
source with the power spectrum depicted in FIG. 2(b). FIG. 2 (a)
thus shows an embodiment of an optical measurement setup 200
comprising a smart phone 202 with a built in LED-light as a light
source 204. The light source 204 is, with the smart phone mounted
on fixture 206, aligned with a spectrometer in the form of a
mini-spectrometer 208 utilized to collect light waves passing
through a sample 210 to be analysed. The sample 210 is kept in a
container 212 in the form of a cuvette placed in a holder 214 that
is mounted on a stand 216. The mini-spectrometer 208 is mounted in
a spectrometer stand 218 with its electronics 220 coupled to a PC
222 or other control or processing device via control and/or data
cable 224.
[0111] The graph in FIG. 2(b) shows the smart phone power spectra
versus wavelength in an example of the experimental setup. In one
example the measured optical power of the light beam exhibited a
maximum power of approximate 35 .mu.W at a wavelength of 623 nm,
and in another example a maximum power of 47 .mu.W at a wavelength
of 615 nm as indicated in the graph of FIG. 2(c). In examples a
mini-spectrometer C11708MA (Hamamatsu/Japan) was used to measure
the light intensity as the light passes through test substances
with spectral response ranging from 640 to 1010 nm. In those
examples the wavelength reproducibility varied between -0.5 to 0.5
nm and a maximum of 20 nm full width at half maximum FWHM spectra,
under constant light conditions. The sample under test was placed
between the, in this example mobile, light source and the
minispectrometer, as described and as shown in FIG. 2(a). The
measurements were conducted with the room lights on. The distances
between the light source, the spectrometer, and the sample holder
were adjusted to eliminate any possible interference and to
stabilize the spectrometer performance.
[0112] FIG. 2(c) illustrates the incident, reflected, and
transmitted light intensities. The spectrometer was aligned with
the light source and a sample cuvette accommodating the sample to
achieve a straight path of light. The light intensities were linked
through the Kirchhoff's Law of Radiation, which correlates the
optical absorbance (A), transmittance (T), and reflection (R) along
with the incident wave (I). In this text, the percentage of the
relative change in light intensity (.DELTA.I.sub.r) is introduced
and defined to be the difference between the two measured peaks
divided by their maximum peak times 100%.
[0113] An experimental setup in accordance with this embodiment may
be used to characterize the two spike proteins subunits, S1 and S2
that are encoded by all coronaviruses and allow virus entry into
susceptible cells, as illustrated in FIG. 1(b). FIG. 3(a) shows an
example of the optical responses for both spike proteins S1 and S2
along with their corresponding two blank samples. The measured
optical intensity changed from 600 to 750 nm, within the light
source spectrum measured earlier as illustrated in FIG. 2(b). The
response of the blank samples was performed first, followed by the
samples with the two protein suspensions, the responses to which
were recorded individually.
[0114] The graphs in FIG. 3 show examples of optical measurements
of samples with the spike protein subunits S1 and S2 in a
microcentrifuge tube, wherein:
[0115] FIG. 3(a) shows measured light intensity over wavelength
responses for spike proteins S1 and S2 at the highest concentration
individually (S1B and S2B, respectively), along with their
corresponding blanks.
[0116] FIG. 3(b) shows time domain measurements of the
corresponding samples, the blank here an empty microcentrifuge tube
shown to the upper left and the S1B sample with water to the upper
right in grey circles, versus the blank again an empty
microcentrifuge tube before S2B to the upper left and the S2B
sample with water to the lower right in red circles, at a
wavelength of 623 nm.
[0117] FIG. 3(c) shows measured optical responses for the mixed
proteins versus time. Samples S1 and S2 were at 5000 copies per ml,
S2C, S2D, S2E and S2F are the serial dilutions of S2B at 10-, 100-,
1,000- and 10,000-fold, respectively.
[0118] FIG. 3(d) shows relative change in light intensity per light
path versus loaded mass. All optical responses were measured at 623
nm. Light intensity was measured as arbitrary units (a.u).
[0119] FIG. 3(a) reveals that S2 exhibited a higher "back
scattering" or absorbance than S1. The response of the two blank
samples was quite comparable, showing the reproducibility of the
results. Since the maximum difference between the blank and the two
protein samples was observed at 623 nm, this wavelength was chosen
for further experimentation to illustrate embodiments, which is
also the wavelength at which the optical power of the smart phone
is at its maximum.
Optimization of the Sample Reading Conditions
[0120] Embodiments comprise calibrating a system for opto-electric
virus detection in order to correct for errors that may occur when
samples are loaded into the sample holder, and the angle and
position of a container for a sample for example in the form of a
microcentrifuge tube changes which may affect the results obtained.
To ensure that the results are reproducible, the measurements for
the same samples may, as in embodiments, be conducted over
different days. In such embodiments the setup may be standardized
for example on each day since the position of the mobile phone,
spectrometer and/or samples may vary. To overcome this caveat and
have more consistence measurements without constant
standardization, advantage may be made of the ability of the
spectrometer to provide light intensity measurements over time.
Hence, after placing the microcentrifuge tube into the holder, the
measurement mode may be started and the corresponding "blank"
recorded. Then the sample would be added after for example
.about.1.5 seconds, while keeping the measurement mode on. FIG.
3(b) illustrates the corresponding measurement profile for the
buffer, in this case water. As would be expected, the blank
exhibited the maximum measured light intensity, while the water
sample showed lower light intensity than the blank. The shift in
response time due to loading of the sample ranged between 1.5 to
2.3 seconds.
Test of the Spike Proteins Using the Experimental Set Up of
Embodiments
[0121] In an example of conducting a test on the proof-of-principle
of these embodiments for experimental design, initially a mixing
experiment was conducted at a light wavelength of 623 nm. Towards
this end, 250 .mu.L of S1 protein solution was tested at the same
maximum concentration at 5,000 copies/mL followed by addition of
same amount of S2. FIG. 3(c) shows the light intensity (as
arbitrary units, a.u.) with time as the protein samples were added
to the container in a sequential manner. This was followed by
addition of 250 .mu.L of ten-fold serial dilutions of the S2
protein at equal time intervals to the S1+S2 samples. As can be
seen from FIG. 3(c), with the addition of the S2 protein, the light
intensity increased. The biggest increase was observed with the
concentrated S2 sample followed by its ten-fold dilution samples
S2A, S2B, S2C, etc., until S2F addition as a 1:10,000 dilution had
no extra effect on the increase in light intensity, indicated the
limit of detection of the assay to be 5000 molecules per
mL.times.250 .mu.l.times.1/10,000=125 molecule per mL. These
results indicated that ratio between the S1 and S2 protein
concentration plays an important role in the light intensity levels
measured. The ratio of S1 and S2 in the virus is the same since
both come from the cleavage of S protein. However, the S1 subunit
is expressed on the cell surface, while the S2 subunit is embedded
in the lipid bilayer of the cell membrane; therefor S2 is less
available at the cells surface, which should affect light intensity
less than S1 despite equal ratios.
[0122] Table 1 below lists the extracted parameters at specific
time points. The relative change in light intensity per light path
length is a constructed parameter that should correlate with the
loaded mass (concentration) of the protein in a suspension.
TABLE-US-00001 TABLE 1 List of measured and extracted parameters.
Light Length of Sample Intensity the Light Mass of Protein
.DELTA.I.sub.r per length description [a.u.] Path [mm] Tested
[.mu.g] [%/mm] S1B 21215 0.11111 1 104 S1B + S2F 21080 0.22222
1.0001 55 S1B + S2E 21265 0.33333 1.0011 34 S1B + S2D 21785 0.44444
1.0111 21 S1B + S2C 23440 0.55556 1.1111 4 S1B + S2B 23875 0.66667
2.1111 0.8
[0123] FIG. 3(d) shows the change in relative light intensity
versus the total mass of the tested samples. As shown in Table 1,
it indicates that as the mass of the protein increased in our
experimental system, the intensity of light also increased,
irrespective of the nature of the protein.
[0124] FIGS. 4(a) and (b) illustrate the definitions of the light
intensities and light path length. The light source, for example a
smart mobile integrated light source, emits a light intensity
(I.sub.0) that is the maximum intensity that can be measured in an
experimental setup in accordance with the above embodiments. The
blank intensity (I.sub.b) is the measured intensity that goes
through the empty container holding the sample such as the
microcentrifuge tube used in the examples above. The instantaneous
intensity (I) is the recorded light when it passes through the
sample. This amount of light intensity strongly depends upon the
buffer in which the sample is solubilized/dissolved in, its
composition, the light path length, the kind of the suspended
analytes and its size in the buffer. The light path length depends
upon the loaded amount of suspension inside the container. The path
length varies from zero up to the container length (L). For a
sample with a specific volume (V), the corresponding path length is
equal to the volume over the cross-sectional area of the container
(A). Equation (1) expresses the relationship between the relative
change in light intensity per light path length and loaded mass
(m), as follows in equation (1):
m=m.sub.i+m.sub.fe.sup.-.alpha.(.DELTA.I/l) (1)
[0125] In equation (1): m.sub.i, m.sub.f and l are the initial mass
of the buffer, the mass of the final suspension composite, and the
light path length, respectively. .alpha. is the decay factor,
unique for each control buffer. Its unit is in mm and could be
correlated with the material absorptivity. .DELTA.I is the relative
changes in light intensity expressed as follows in equation
(2):
.DELTA.I=(1-I/I.sub.b).times.100% (2)
[0126] In equation (2): I and I.sub.b are the instantaneous
measured light intensity of the suspension and the corresponding
blank, respectively. FIG. 4(c) shows the relationship between mass
and the relative change per length after fitting the measured
points with the exponential function. As can be seen, with more
sample volume the path length increases and light intensity
decreases, hence relative change decrease dramatically.
[0127] In method and system embodiments, relations for example
those described herein, above and/or below, are applied to
determine concentration of virus or virus load by serial dilution
of antibody and simultaneous measurements using one or more
microfluidic channels filled with a virus sample in solution. A
microfluidic sensing device comprised in embodiments comprises one
or more microfluidic channels. In the case with a plurality of
microfluidic channels, the channels are in preferred embodiments
arranged in parallel.
[0128] A light source may as in some embodiments integrated with
the microfluidic sensing device, or the microfluidic sensing device
may as in other embodiments be configured to receive light from an
external light source. Both variant embodiments are configured such
that light passes through the sample in each of the one or more
microchannels, or groups of microchannels.
[0129] Embodiments of such microfluidic sensing devices further
comprise an electric field device configured to apply an electric
field over individual microchannels or groups of microchannels.
Such an electrical field device may be integrated in the
microstructure of the microfluidic device or be configured as a
device external to the microstructure of the microfluidic device.
The electrical field device may comprise electrodes and/or
electrode connectors configured for individual channels, groups of
channels or the whole microstructure.
[0130] Embodiments of such a microfluidic sensing device comprises
a light sensor and/or an electrical sensor. Such sensors may be
integrated in the said one or more channels or in the
microstructure surrounding the channels, and said sensors are
configured such that they can measure light and/or electrical
parameters, e.g., scattering parameters, for each individual
microchannel or groups of microchannels.
[0131] Mathematical models based on such said relations are used to
extract a measurement value for the concentration of virus or virus
load in the sample, thus reflecting the concentration of virus or
virus load of test subject or patient from which the sample is
taken. Quantification of the measurement value is for example
carried out by means of calibration values e.g., represented in
curves or tables. Such calibration values can for example be
generated by calibrating parameter values determined in accordance
with embodiments herein with known values for virus concentration
or virus load. In embodiments, a quantification of concentration of
virus concentration or virus load is determined from the
differences of capacitance voltage profiles, and by processing a
difference signal using semiconductor theory to extract single
parameters or a collection of multiple parameters to determine said
virus concentration or virus load.
[0132] Variants of these microfluidic channel embodiments, as with
other embodiments herein, are configured to use for the detection
of for SARS-CoV-2 diagnosis, whereas other variants are configured,
dependent on selection of the antibody used, for detection of any
respiratory virus such as influenza, respiratory syncytial virus
(RSV), adenoviruses, or other coronaviruses like SARS and MERS.
[0133] Method embodiments for these purposes are configured such
that: a said sample is placed and distributed in one or more
microfluidic channels; measurement of said light scattering
parameters and/or said electrical parameters is conducted for the
sample content in each of said microfluidic channels; and virus
concentration and/or virus load is determined based on said
measured parameters.
[0134] In further variants of such embodiments said sample is
diluted such that there is stepwise increasing dilution of antibody
content in said one or more microfluidic channels and said
measurement of parameters is conducted for said dilution steps.
[0135] In method embodiments said sample is placed and distributed
in a plurality of parallel microfluidic channels; the antibody
content is serially diluted in said plurality of parallel channels,
simultaneously measuring said parameters in said plurality of
channels; and said virus concentration and/or virus load is
determined based on said measured parameters.
[0136] The method of the preceding claim 30, wherein the virus
concentration and/or virus load is based on a mathematical
relationship between said parameters and virus concentration and/or
virus load, said mathematical relationship for example being
calibrated against known virus concentration or virus load.
[0137] System embodiments for the above purposes, comprise: a
microfluidic sensing device with one or more one or more
microfluidic channels configured for placing and distributing a
said sample in one or more of said microfluidic channels; one or
more sensors configured for measurement of said light scattering
parameters and/or said electrical parameters for the sample content
in each of said microfluidic channels; and code portions, in said
processing device, configured to determine virus concentration
and/or virus load based on said measured parameters.
[0138] Further variants of system embodiments are configured for
dilution of said sample such that there is stepwise increasing
dilution of antibody content in said one or more microfluidic
channels and said measurement of parameters is conducted for said
dilution steps.
[0139] In further system embodiments: said microfluidic sensing
device is configured with a plurality of parallel microfluidic
channels for placing and distributing said sample; said
microfluidic sensing device is configured for serially diluting the
antibody content in said plurality of parallel channels,
simultaneously measuring said parameters in said plurality of
channels; and said system is configured to determine said virus
concentration and/or virus load based on said measured
parameters.
[0140] In some such system embodiments, said code portions are
configured such that the virus concentration and/or virus load is
based on a mathematical relationship between said parameters and
virus concentration and/or virus load, said mathematical
relationship for example being calibrated against known virus
concentration or virus load.
[0141] These embodiments with microfluidic channels may be applied
in conjunction with other embodiments described herein or
independently.
[0142] Above it is described by means of exemplifying embodiments
how to detect proteins in solution using light. Further, as in
embodiments described herein, light intensity is used to
characterize the binding interactions of the spike protein with the
viral receptor ACE2. As an exemplifying demonstration of this
mechanism, two different variants of the S1 subunit of the spike
protein, S1A(S1X) and S1B(S1Y), were tested. One of these variants
could bind ACE2 with a much stronger affinity than the other one.
The S1 subunit of the spike protein S1A and S1B were tested along
with a non-specific control protein, bovine serum albumin (BSA)
that should not bind to ACE2. These proteins were selected to
demonstrate the detection of the binding process with ACE2 over
time. Examples of measurement results are shown in FIG. 5 with
graphs of optical detection of binding interactions between ACE2
and other proteins. FIG. 5(a) shows measured light intensities over
time for individual assessment of ACE2, S1A, SIB, and BSA. FIG.
5(b) shows the measured mixed light intensities versus time for
ACE2 mixed with either S1A, or S1B, or BSA. FIG. 5(c) shows the
measured ACE2-S1A interaction profile for an extended time period.
FIG. 5 (d) shows extracted slopes for the individual and mixed
protein suspensions.
[0143] The measurement process in this example started with the
blank, and after 200 seconds, 250 .mu.L of ACE2 protein suspension
was tested, as depicted in the graph of FIG. 5(a). This process was
repeated for S1A (S1X), S1B (S1Y), and BSA and their responses to
light were measured individually in the same manner as ACE2.
[0144] The corresponding individual profiles of ACE2, S1A (S1X),
S1B (S1Y) and BSA are depicted in FIG. 5(a) which showed a straight
constant line over time. The responses of the various protein
mixtures were read over a period of 15 minutes and are shown in
FIG. 5(b). This is interpreted to mean that there was no
protein-protein interaction if the line was straight and constant,
otherwise protein-protein interaction occurs.
[0145] To explore the interaction and binding characteristics, by
example between ACE2 and S1A (S1X) in more detail, the measurement
time between the two proteins was extended over one hour the
results of which are plotted in FIG. 5(c). As can be seen, a nice
"hump" was observed as an increase in arbitrary units with time
that was not observed in the other protein mixtures tested. The
corresponding intensity of the interaction (I.sub.ia) versus time
(t) was fitted with a quartic polynomial as follows in equation
(3):
I.sub.ia=a+bt+ct.sup.2+dt.sup.3+et.sup.4 (3)
[0146] In embodiments the following coefficients in equation (3)
have the values: a, b, c, d and e are 20215.00393.+-.6.54838,
-0.15559.+-.9.83118.times.10.sup.-4,
1.70666.times.10.sup.-5.+-.5.093.times.10.sup.-8,
-4.41996.times.10.sup.-10.+-.1.09328.times.10.sup.-12 and
3.46019.times.10.sup.-15.+-.8.30214.times.10.sup.-18, respectively.
These coefficients can be used in embodiments to detect and
identify the kind of analyte that binds with the ACE2 receptors.
With other antigens and elements, the coefficients would have
values characterised by the specific antigens or elements.
[0147] FIG. 5(d) shows the corresponding slopes that represent the
change of the light intensities over time. Next, each protein was
mixed with, in this example, the ACE2 antigen separately to detect
any possible binding effect. The measurements started with first
loading the ACE2 in the blank container, then after 200 ms, each
protein was added to the ACE2. The ACE2+BSA and ACE2+S1B (S1Y)
responses exhibited almost constant lines with corresponding
responses shown in FIG. 5(d), suggesting highly reduced or lack of
any interaction as observed when the proteins were tested
individually. However, the ACE2+S1A (S1X) profile showed a linear
straight line with the maximum-recorded slope. The corresponding
light intensity line increased over time, suggesting an interaction
between the S1A (SIX) protein and the ACE2 receptor. It is
noticeable that the slopes of the mixed proteins with ACE2 that did
not exhibit much interaction, i.e., S1B (S1Y) and BSA, their slopes
after mixing were less than the sum of their individual slopes. On
the other hand, the ACE2+S1A (S1X) slope was higher than the sum of
the individual ACE2 plus S1A (S1X) slopes, revealing a synergistic
effect on light intensity. Based on these observations, our results
suggest that the S1A (S1X) protein exhibits stronger interactions
with ACE2, while BSA and S1B (S1Y) had weaker interactions with
ACE2. These observations are confirmed by the fact that whereas S1A
(S1X) has a higher affinity for ACE2 (for example 2 .mu.g/mL S1A
can bind 1.5-15 ng/mL ACE2), S1B (S1Y) reportedly has a much lower
affinity (for example 2 .mu.g/mL S1B binds 0.5-8.7 ng/mL ACE2), as
tested in enzyme-linked immunosorbent assays (ELISA) by the company
that synthesized these proteins (ProSci, USA).
[0148] In further embodiments, the described optical system is used
to detect protein-protein interactions. Using proteins that are
well known to interact with each other in such measurements confirm
the results described above. In an example, this can be done by
testing the molecular interactions between an antigen and an
antibody which is similar to the interaction between the spike
protein and its receptor. In such an example, two proteins were
tested along with their specific antibodies: the first protein was
the receptor binding domain RBD of SARS-CoV-2 spike protein and its
antibody and the other was the nucleocapsid protein NCP of
SARS-CoV-2 and its antibody. Similar to the procedure described
above, the two proteins were tested individually in an optical
assay as described above followed by addition of their
corresponding antibodies that were mixed and their interactions.
FIG. 6 shows graphs of optical detection of the binding affinities
between proteins and antibodies in this example. FIG. 6(a) shows
the binding between the receptor binding domain RBD, in this
example, of SARS-CoV-2 spike protein and its antibody AB. Upon the
addition of the antibody, marked Loading in FIG. 6(a) an
interaction peak was recorded as indicated with a circle FIG.
6(a).
[0149] FIG. 6(b) shows the binding between, in this example, the
nucleocapsid protein NCP of SARS-CoV-2 and its antibody AB. As
indicated in the graph, the measurement started with a blank, then
with added NCP and after that with added antibody NCP+AB three
times, indicated NCP+AB, NCP+2AB and NCP+3AB, respectively. The
antibody was added a second time to NCP since no binding
interaction was observed the first time. After the second addition
of antibodies 2AB a peak in the light intensity indicating binding
activity was observed, as indicated with a circle in FIG. 6(b). To
further confirm the result, the antibody was added a third time
NCP+3AB, and this time once again, the binding interaction was not
apparent. In this exemplifying case, the conclusion is that the
binding effect occurred at specific antibody concentration. For a
virus-based suspension, embodiments therefore use a fixed antibody
concentration and serially dilute the virus suspensions and conduct
the binding measurements. At a specific virus concentration,
binding effect will appear on the optical response.
[0150] FIGS. 6(c) and (d) illustrate the corresponding optical
responses for, in this example, NC protein and its corresponding
antibody when they were mixed either inside (FIG. 6(c)) or outside
(FIG. 6(d)) the microcentrifuge tube, respectively. Peaks, or
humps, in the light intensity indicating binding activities are
marked with circles in the graphs. Inside mixing means that the
protein was added to the tube and the antibody was added after 100
seconds, while in the outside mixing scenario, both the protein and
antibody were mixed prior to being loaded in the tube for optical
measurements. As can be seen, the binding response could be
detected in each case in the form of the appearance of the hump.
However, this "hump" was a lot more pronounced when the protein and
the antibody were mixed prior to test testing than when they were
added sequentially. When employing embodiments in real life
scenarios with patient samples, the antibody should be already
bound to the viral or bacterial antigen at the time of detection.
Preferably a potentially virus containing sample from a patient
should be mixed with antibody before optical detection
measurement.
[0151] Embodiments makes use of the effect of direct current DC
biasing on the ability of two proteins to bind specifically. An
example to illustrate this was carried out by subjecting the
nucleocapsid protein (NCP or NC protein) solution to DC voltage
bias. FIG. 7 shows graphs of Opto-electrical measurements of
nucleocapsid protein (NC protein) with direct current DC biasing.
FIG. 7(a) shows an example of measured NC protein optical response
versus time at different DC bias voltages. An applied bias should
result in an induction of current across the suspension. If this
current is high enough, it should have the potential to destroy the
protein physiology and functionality, resulting in the loss of
specific protein-protein interactions. In this example, the NC
protein solution was loaded in an electroporation cuvette (rather
than a microcentrifuge tube as in previous examples) that
incorporates two electrodes with a volume of 0.5 mL and a
separation distance of 0.4 cm. This should result in a breakdown
electric field of 7.5 V/cm. At this field onwards, the binding
between the protein and the antibody should be affected. Above this
field the sample will be incapacitated. As shown in FIG. 7(a), the
optical response decays slowly with the application of DC bias. At
3 volts DC bias, the optical response decays with a considerable
step, and increasing the DC bias further should burn the suspension
and destroy it.
[0152] The breakdown field depends on the electrical
characteristics of both the buffer and the analyte such as
proteins, viruses, etc. In further exemplifying application of
embodiments, the suspension of protein was subjected to 3 V for 1
minute and then the antibody to nucleocapsid NC was added to the NC
protein solution. The corresponding measured response is shown in
FIG. 7(b). The measured response was observed to be noisy and did
not show a clear binding effect when compared with FIG. 6(c) that
shows the optical response for the same protein and antibody
without the application of DC bias. Embodiments are configured to
create a corresponding vaccine for a disease by subjecting its
corresponding virus to DC bias which will affect its infectivity
and destroy its physiology and communicability. Furthermore,
embodiments of the optical detection in time domain are configured
to be used for monitoring and detecting the efficiency of vaccine
process development.
[0153] Embodiments are configured for virus detection on samples on
paperbased nitrocellulose membrane. The nitrocellulose membrane is
a popular matrix that is frequently used due to its high
protein-binding affinity with a pore size of 0.25-0.45 .mu.m in
paper-based diagnostics. Protein molecules usually bind to the
nitrocellulose membranes through hydrophobic interactions. Due to
the ease of their handling, cheap cost, and the presence of
hydrophobic interactions between them and the suspended proteins,
we tested whether the binding between the SARS-CoV-2 spike protein
and antibody could be detected optically when both were added to
each other on the nitrocellulose membrane. Using embodiments
illustrated with the experimental setup described in FIG. 2(a), the
optical responses for nitrocellulose membrane, nitrocellulose
membrane and spike protein alone, nitrocellulose membrane and
antibody against spike protein alone, and nitrocellulose membrane
spike protein-antibody were measured in illustrating example. FIG.
8 shows graphs of this illustrating example of protein-protein
interaction measurements on paperbased nitrocellulose membrane
(NM).
[0154] FIG. 8(a) shows optical responses on nitrocellulose membrane
(NM) alone, nitrocellulose membrane and spike protein (NM+P), and
nitrocellulose membrane and antibody to spike protein (NM+AB)
alone. The graph in FIG. 8(a) shows that both the antibody alone
and spike protein alone exhibited higher light intensity than the
nitrocellulose membrane alone with almost a straight line with
constant slope over a time period of 10 seconds. The on-paper
measured optical responses exhibited fluctuations as in the samples
measured using microcentrifuge tubes. This implies that these
fluctuations are not due to any kind of interactions, but instead
are due to the spectrometer conversion process. FIG. 8(b) summaries
the interaction measurements which start with the membrane NM.
After 100 mSecond, the antibody suspension AB was loaded on the
membrane and measurements were conducted up to 1000 mSecond. Next,
the spike protein sample was loaded AB+Protein and measurements
were continued up to 5000 mSecond. As shown in FIG. 8(b), the
interaction peaks appeared clearly within the indicating circle. It
is worth noting that the membrane size, shape, and charge of
biomolecules, pH and viscosity of the control buffer, as well as
the composition influences the corresponding optical response and
binding interactions.
[0155] As these illustrating protein-antibody interactions took
place within a time domain, the measurements are further
illustrated using the well-known technique of chronoamperometry to
detect this interaction electrically. Chronoamperometry is an
electrochemical technique in which an electric potential is applied
between two electrodes to measure the resulting current at the
surface that is created from faradaic processes over time. The
corresponding chronoamperometry measurements were conducted
accordingly as plotted in FIG. 9. FIG. 9 thus shows a graph of
chronoamperometry measurements indicating current versus time for
different sample suspensions, in accordance with embodiments. In
FIG. 9, the lowest curve shows protein P (NCP), the second lowest
curve shows antibody AB, whereas the next to upper curve shows a
first measurement on protein-antibody suspension ABNCP1 and the
uppermost curve shows a second measurement on protein-antibody
suspension ABNCP2. The two curves ABNCP1 and ABNCP2 pertain to two
different measurements that uses the same nucleoprotein and the
same antibody.
[0156] The applied potential in this example was of 0.5 mVolts, the
minimum voltage that can be applied without causing any harm to the
protein and antibody; therefore, it should not have affected the
interaction process. As shown in FIG. 9, the electrical current
measurements for the protein-antibody suspensions ABNCP1 and ABNCP2
exhibited exponential growth profiles, whereas the individual
profiles for protein P (NCP) and antibody AB did not exhibit such
behaviour. These embodiments can be used in a detection strategy.
As shown in FIG. 9, the binding took place between 50 and 250
seconds, a period during which protein-antibody solutions ABNCP1
and ABNCP2 (ABP) exhibited higher measured electrical current
values compared with protein P or antibody AB currents alone.
[0157] As described and illustrated in the above examples,
different embodiments are configured as follows.
[0158] Specific examples of device and method have been described
herein for purposes of illustration. These are only examples. The
technology provided herein can be applied to device and method
other than the examples described above. Many alterations,
modifications, additions, omissions and permutations are possible
within the practice of this invention. This invention includes
variations on described embodiments that would be apparent to the
skilled addressee, including variations obtained by: replacing
features, elements and/or acts with equivalent features, elements
and/or acts; mixing and matching of features, elements and/or acts
from different embodiments; combining features, elements and/or
acts from embodiments as described herein with features, elements
and/or acts of other technology; and/or omitting combining
features, elements and/or acts from described embodiments.
* * * * *
References